Functional Specialization ofChlamydomonas reinhardtiiCytosolic Thioredoxin h1 in the Response to Alkylation-Induced DNA Damage

Abstract

ABSTRACTDNA damage occurs as a by-product of intrinsic cellular processes, like DNA replication, or as a consequence of exposure to genotoxic agents. Organisms have evolved multiple mechanisms to avoid, tolerate, or repair DNA lesions. To gain insight into these processes, we have isolated mutants hypersensitive to DNA-damaging agents in the green algaChlamydomonas reinhardtii. One mutant, Ble-1, showed decreased survival when it was treated with methyl methanesulfonate (MMS), bleomycin, or hydrogen peroxide (H2O2) but behaved like the wild type when it was exposed to UVC irradiation. Ble-1 carries an extensive chromosomal deletion that includes the gene encoding cytosolic thioredoxin h1 (Trxh1). Transformation of Ble-1 with a wild-type copy ofTrxh1fully corrected the MMS hypersensitivity and partly restored the tolerance to bleomycin.Trxh1also complemented a defect in the repair of MMS-induced DNA strand breaks and alkali-labile sites. In addition, a Trxh1-β-glucuronidase fusion protein translocated to the nucleus in response to treatment with MMS. However, somewhat surprisingly,Trxh1failed to correct the Ble-1 hypersensitivity to H2O2. Moreover,Trxh1suppression by RNA interference in a wild-type strain resulted in enhanced sensitivity to MMS and DNA repair defects but no increased cytotoxicity to H2O2. Thioredoxins have been implicated in oxidative-stress responses in many organisms. Yet our results indicate a specific role ofChlamydomonasTrxh1 in the repair of MMS-induced DNA damage, whereas it is dispensable for the response to H2O2. These observations also suggest functional specialization among cytosolic thioredoxins since anotherChlamydomonasisoform (Trxh2) does not compensate for the lack of Trxh1.